Abstract
2-Oxoglutarate dehydrogenase (Ogdh) is an important mitochondria redox sensor that can undergo S-glutathionylation following an increase in H2O2 levels. Although S-glutathionylation is required to protect Ogdh from irreversible oxidation while simultaneously modulating its activity it remains unknown if glutathione can also modulate reactive oxygen species (ROS) production by the complex. We report that reduced (GSH) and oxidized (GSSG) glutathione control O2∙-/H2O2 formation by Ogdh through protein S-glutathionylation reactions. GSSG (1mM) induced a modest decrease in Ogdh activity which was associated with a significant decrease in O2∙-/H2O2 formation. GSH had the opposite effect, amplifying O2∙-/H2O2 formation by Ogdh. Incubation of purified Ogdh in 2.5mM GSH led to significant increase in O2∙-/H2O2 formation which also lowered NADH production. Inclusion of enzymatically active glutaredoxin-2 (Grx2) in reaction mixtures reversed the GSH-mediated amplification of O2∙-/H2O2 formation. Similarly pre-incubation of permeabilized liver mitochondria from mouse depleted of GSH showed an approximately ~3.5-fold increase in Ogdh-mediated O2∙-/H2O2 production that was matched by a significant decrease in NADH formation which could be reversed by Grx2. Taken together, our results demonstrate GSH and GSSG modulate ROS production by Ogdh through S-glutathionylation of different subunits. This is also the first demonstration that GSH can work in the opposite direction in mitochondria-amplifying ROS formation instead of quenching it. We propose that this regulatory mechanism is required to modulate ROS emission from Ogdh in response to variations in glutathione redox buffering capacity.
Highlights
It is appreciated more than ever that controlled low grade reactive oxygen species (ROS) formation is required to modulate cell functions in tandem with other signaling cascades [1]
Purified Oxoglutarate dehydrogenase (Ogdh) from porcine heart (α-ketoglutarate dehydrogenase; Sigma Catalog Number K1502), Grx2, mannitol, sucrose, EGTA, and Hepes, 2-oxoglutarate, NAD þ, NADH, NADPH, glutathione reductase (GR), GSH, GSSG, hydroxyethyl disulfide (HEDS), thiamine pyrophosphate (TPP), Coenzyme A (CoASH), 3-methyl-2-oxopentanoic acid (KMV) and horseradish peroxidase (HRP), superoxide dismutase (SOD), catalase (CAT), alamethicin, 1-chloro-2,4-dinitrobenzene (CDNB), and defatted BSA were purchased from Sigma
To ascertain if Ogdh was (1) sensitive to S-glutathionylation by GSSG and (2) if GSSG induced S-glutathionylation modulates O2∙−/hydrogen peroxide (H2O2) formation by Ogdh, a series of experiments were carried out to examine if S-glutathionylation can control ROS production by Ogdh
Summary
It is appreciated more than ever that controlled low grade ROS formation is required to modulate cell functions in tandem with other signaling cascades [1]. Mitochondria contain a number of S-glutathionylation targets which includes enzymes and respiratory complexes that generate ROS [8] These reactions are highly specific and enzymatically mediated. Grx, which is found in the matrix of mitochondria, catalyzes the reversible S-glutathionylation of Complex I in response to changes in GSH/GSSG which modulates ROS production [15,16]. Pyruvate dehydrogenase (Pdh), an enzyme similar in structure to Ogdh which commits carbohydrate degradation product pyruvate to Krebs cycle metabolism, serves as a redox sensor, increasing and decreasing ROS production in response to changes in mitochondrial redox buffering capacity [23].
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